Novel gold nanoparticle aggregates and their applications

ABSTRACT

The invention is drawn to novel gold nanoparticles that are used in a dual optical method for sensitive and selective detection of antigens. The gold nanoparticle aggregates are synthesized from gold hydrochloride and sulfur salts in an aqueous solution. The aggregates can be selectively sized using a spectral notch filter that results in an improved product with versatile uses. The gold nanoparticles can also be used in improved optical communications devices.

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 60/667,151 entitled “Novel Gold NanoparticleAggregates and Their Applications”, filed Mar. 30, 2005, and U.S.Provisional Patent Application Ser. No. 60/711,808 entitled “Novel GoldNanoparticle Aggregates and Their Applications”, filed Aug. 26, 2005,which are herein incorporated by reference in their entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to particles comprising a metallic coreand sulfur species on their surface with useful properties. Theinvention further relates to methods of using the particles fordetecting chemical and biological analytes, and in use in opticalcommunications.

BACKGROUND

During the 1980s Raman Scattering in fibers was demonstrated by Lin,Stolen, and other co-workers of AT&T Bell Laboratories in Holmdel, N.J.using Raman lasers operating between 0.3 to 2.0 μm. In the early yearsof the Raman fiber before extensive work had begun, no one perceivedthat a Raman fiber could be pumped by a practical semiconductorlaser-based source or that an efficient CW-pumped Raman Fiber Laser waspossible. However, with the development of Cladding-pumped Fiber Lasersand Fiber Bragg Gratings, diode-laser-based CW Raman Fiber Lasers havebeen made efficient, emitting at various wavelengths throughout theinfrared spectrum a reality. (See van Gisbergen et al. (1996) Chem.Phys. Lett. 259: 599-604.)

Raman spectroscopy is a powerful optical technique for detecting andanalyzing molecules. Its principle is based on detecting light scatteredoff a molecule that is shifted in energy with respect to the incidentlight. The shift, called Raman shift, is characteristic of individualmolecules, reflecting their vibrational frequencies that are likefingerprints of molecules. As a result, the key advantage of Ramanspectroscopy is its molecular specificity while its main limitation isthe small signal due to low quantum yield of Raman scattering. One wayto enhance the Raman signal is to tune the excitation wavelength to beon resonance with an electronic transition, so called resonance Ramanscattering. This can usually produce an enhancement on the order of10²-10³.

Another technique to enhance Raman scattering is surface enhancement byroughened metal surfaces, notably silver and gold, that provides anenhancement factor on the order of 10⁶-10⁸. This is termed surfaceenhanced Raman spectroscopy (SERS). Similar or somewhat largerenhancement factors (˜10⁸-10¹⁰) have been observed for metal, mostlysilver or gold, nanoparticles.

In the last few years, it has been shown that an even larger enhancement(˜10¹⁰-10¹⁵) is possible for aggregates of metal nanoparticles (MNPs),silver and gold. The largest enhancement factor of 10¹⁴-10¹⁵ has beenreported for rhodamine 6G (R6G) on single silver nanoparticleaggregates. This huge enhancement is thought to be mainly due tosignificant enhancement of the local electromagnetic field of thenanoparticle aggregate that strongly absorbs the incident excitationlight for the Raman scattering process. With such large enhancement,many important molecules that are difficult to detect with Ramannormally can now be easily detected. This opens many interesting and newopportunities for detecting and analyzing molecules using SERS withextremely high sensitivity and molecular specificity.

SERS can also be developed into a molecular imaging technique forbiomedical and other applications. Existing Raman imaging equipmentshould be usable for SERS imaging. SERS will provide a much-enhancedsignal and thereby significantly shortened data acquisition time, makingthe technique practically useful for medical or other commercial andindustrial applications including chip inspection or chemicalmonitoring. SERS is also useful for detecting other cancer biomarkersthat can interact or bind to the MNP surface. For example, Sutphen etal. have recently shown that lysophospholipids (LPL) are potentialbiomarkers of ovarian cancer (Sutphen et al. (2004) Cancer Epidemiol.Biomarker Prev. 13: 1185-1191).

For many practical applications, for example SERS and optical filters,it is highly desirable to narrow the distribution of size/shape ofnanoparticle aggregates. For SERS in particular, the incident light hasto be on resonance with the substrate absorption. Only thosenanoparticle aggregates that have resonance absorption of the incidentlight are expected to be SERS active. It is thus extremely beneficial tohave a narrow size/shape distribution and thereby narrow opticalabsorption.

Fluorescent nanoparticles (quantum dots (QDs) such as semiconductorquantum dots, SQDs) have been used recently as fluorescent biologicalmarkers and have been found to be extremely effective. They offeradvantages including higher stability, stronger fluorescence, tunabilityof color, and possibility of optical encoding based on different sizedor colored SQDs.

A method of synthesis for gold nanoparticle aggregates (GNAs) has beendescribed in the prior art (see Norman et al. (2002) J. Phys. Chem. B,106: 7005-7012). Norman used Na₂S and HAuCl₄ (chloroauric acid). Normansuggested that the product of the reaction is elemental sulfur,elemental gold, free protons, and free chlorine ions. This contrastswith the alternative dogma that the aggregates comprise an Au₂S coreenveloped by an Au shell. Therefore Norman concluded that the reactionproduces aggregates of gold nanoparticles having amorphous sulfur ontheir surface.

Metal nanoparticles have been recognized for their unique opticalproperties that could be exploited in optoelectronic devices.Nanoparticle systems composed of gold, for example, have distinctoptical properties that make them amenable to study by Raman scattering.The Raman spectrum of the adsorbed species is significantly enhanced by10 to 15 orders of magnitude when the metal nanoparticles haveaggregated, leading to enhanced electromagnetic field effects near thesurface that increases the Raman scattering intensity. The greatersensitivity found in the surface enhanced Raman spectroscopy (SERS) ofmetal nanoparticle aggregates facilitates the detection and analysis ofa whole host of molecules that were previously difficult to study.

Wang et al. disclose a method of using SQDs (dye-conjugated CdTenanoparticles, CT-NPs) to detect interactive binding between Ag-CT-NPsand Ab-CT-NPs (Wang et al. (2002) NanoLett. 2: 817-822). Theinteractions were determined by differential quenching or enhancementfluorescence activity of two different sized SQDs (red or green)measured during the analysis.

The chemical methods used historically for the production of goldnanoparticle aggregates (GNAs) results in a wide distribution ofaggregate size. This distribution leads to a broadened absorptionspectrum. Accordingly, researchers have attempted to narrow thelineshape of the spectral peak due to the aggregates by homogenizing thesize of the GNAs after they have been produced. By eliminating certainranges of aggregate size, absorption spectrum peaks should narrowappreciably and concomitantly increase in intensity, resulting in moresensitive detection. Previous attempts to select for a narrow size rangeof aggregates have employed mechanical techniques such as passing asolution of aggregates through a filter. For example, Emory & Nie haveemployed size-selective fractionation using membrane filters to selectfor optically active silver nanoparticles (Emory and Nie, (1997) J.Phys. Chem. B, 102: 493-497).

The use of SERS for analyte detection of biomolecules has beenpreviously studied. U.S. Pat. No. 6,699,724 to West et al. describes achemical sensing device and method (nanoshell-modified ELISA technique)based on the enzyme-linked immunoadsorbant assay (ELISA). The chemicalsensing device can comprise a core comprising gold sulfide and a surfacecapable of inducing surface enhanced Raman scattering (SERS). In much ofthe patent disclosure, the nanoparticle is disclosed as having a silicacore and a gold shell. The patent discloses that an enhancement of600,000-fold (6×10⁵) in the Raman signal using conjugatedmercaptoaniline was observed.

In the nanoshel-modified ELISA technique, antibodies are directly boundto the metal nanoshells. Raman spectra are taken of theantibody-nanoshell conjugates before and after the addition of a samplecontaining a possible antigen, and binding of antigen to antibody isexpected to cause a detectable shift in the spectra.

The conjugation of quantum dots to antibodies used for ultrasensitivenonisotopic detection for use in biological assays has also beenstudied. U.S. Pat. No. 6,468,808 B1 to Nie et al. disclosed an antibodyis conjugated to a water-soluble quantum dot. The binding of the quantumdot-antibody conjugate to a targeted protein will result inagglutination, which can be detected using an epi-fluorescencemicroscope. In addition, Nie et al. described a system in which aquantum dot is attached to one end of an oligonucleotide and a quenchingmoiety is attached to the other. The preferred quenching moiety in theNie patent is a nonfluorescent organic chromophore such as4-[4′-dimethylaminophenylazo]benzoic acid (DABCYL).

Raman amplifiers are also expected to be used globally as a key devicein next-generation optical communications, for example, inwavelength-division-multiplexing (WDM) transmission systems. Ramanscattering occurs when an atom absorbs a photon and another photon of adifferent energy is released. The energy difference excites the atom andcauses it to release a photon with low energy; therefore, more lightenergy is transferred to the photons in the light path.

FIG. 6 shown how the Raman amplifier operates. The Raman amplificationprocess begins as a seed beam (incoming light) passes through theoptical fiber. While it is traveling, a stronger pump beam is releasedfrom another light source and is deflected using a refractive material,such as a mirror. The pump beam and seed beam then come in contact witheach other and the seed beam depletes the energy of the pump beam;therefore the intensity of the light increases and the signal isamplified. Now the signal is capable of traveling long distances, forexample, more than 70 km, without losing a signal. (See, for example,U.S. Pat. No. 6,292,288; Vinson and Webb (2001) Light Amplification: TheFuture Of Optical Communications, Optical Engineering UCSC, SummerVentures of Science and Math, 2001, 7 pp.)

There is therefore a need in the art for use in the biomedicalanalytical industries and the optical communications industries toprovide more sensitive compositions and devices that are inexpensive tomanufacture and easy to use.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a chemical sensor comprising a pluralityof particles, each particle comprising: a core, a shell having at leastone surface and having contact with the core and wherein the shellcomprises a sulfur-oxygen molecular species, and wherein the particlehas been selectively sized using a notch filter and electromagneticradiation, the electromagnetic radiation having a spectral wavelength ofbetween about 350 nm and about 1075 nm. In one embodiment the particlehas a size in the range of about 60 and 200 nm. In another embodimentthe particle has a size selected from the range of between about 60 and150 nm, between about 60 and 100 nm, between about 60 and 80 nm, betweenabout 80 and 200 nm, between about 80 and 150 nm, between about 80 and100 nm, between about 100 and 200 nm, between about 100 and 150 nm, andbetween about 150 and 200 nm.

In a preferred embodiment the core comprises a metal selected from thegroup consisting of gold, silver, platinum, copper, aluminum, palladium,cadmium, iridium, and rhodium. In a more preferred embodiment the corecomprises gold.

In another preferred embodiment the electromagnetic radiation has aspectral wavelength of between about 350 nm and about 650 nm and betweenabout 950 nm and about 1075 nm. In yet a more preferred embodiment theelectromagnetic radiation has a spectral wavelength of between about 350nm and about 775 nm and between about 875 nm and about 1075 nm.

In another embodiment, the chemical sensor comprises a shell thatfurther comprises a linker molecule, the linker molecule selected fromthe group consisting of a thiol group, a sulfide group, a phosphategroup, a sulfate group, a cyano group, a piperidine group, an Fmocgroup, and a Boc group.

In yet a further embodiment, the chemical sensor comprises a support. Ina preferred embodiment, the support comprises a medium that is permeableto an analyte of interest.

In another preferred embodiment, the chemical sensor has a surfacewherein the surface can induce surface enhanced Raman scattering (SERS).

In still another preferred embodiment, the chemical sensor furthercomprises at least one detecting molecule, wherein the detectingmolecule is bound to the surface. In a more preferred embodiment thedetecting molecule is selected from the group consisting of proteins,peptides, antibodies, antigens, nucleic acids, peptide nucleic acids,sugars, lipids, glycophosphoinositols, and lipopolysaccharides.

In a yet more preferred embodiment the detecting molecule is anantibody. In another preferred embodiment, the detecting molecule is anantigen.

In another embodiment, the invention provides a chemical sensor furthercomprising at least one semiconductor quantum dot. In a preferredembodiment the semiconductor quantum dot further comprises a linkermolecule, the linker molecule selected from the group consisting of athiol group, a sulfide group, a phosphate group, a sulfate group, acyano group, a piperidine group, an Fmoc group, and a Boc group.

In a still further embodiment, the invention provides a chemical sensorcomprising at least one semiconductor quantum dot wherein thesemiconductor quantum dot further comprises a detecting molecule,wherein the detecting molecule is bound to the semiconductor quantumdot. In a more preferred embodiment, the detecting molecule is selectedfrom the group consisting of proteins, peptides, antibodies, antigens,nucleic acids, peptide nucleic acids, sugars, lipids,glycophosphoinositols, and lipopolysaccharides.

In a more preferred embodiment, the detecting molecule is an antibody.In the alternative, a more preferred embodiment comprises a chemicalsensing device wherein the detecting molecule is an antigen.

Another embodiment of the invention provides a method for detecting ananalyte in a sample using a chemical sensor, the method comprising thesteps of: i) providing a sample; ii) providing a semiconductor quantumdot comprising a linker molecule (LM-SQD); iii) conjugating the analytein the sample with the LM-SQD thereby producing an analyte-LM-SQDconjugate; iv) providing a chemical sensor comprising a plurality ofparticles, each particle comprising: a core, a shell having at least onesurface and having contact with the core and wherein the shell comprisesa sulfur-oxygen molecular species, and wherein the particle has beenselectively sized using a notch filter and electromagnetic radiation,the electromagnetic radiation having a spectral wavelength of betweenabout 350 nm and about 1075 nm, the shell surface further comprising adetecting molecule; v) incubating the analyte-LM-SQD conjugate with thechemical sensor for a predetermined time period; and vi) measuring theextent of binding between the analyte-LM-SQD conjugate and the chemicalsensor; thereby detecting the analyte in the sample.

In a preferred embodiment the invention provides a method for detectingan analyte that is an ovarian cancer marker antibody. In one embodimentof the invention the detecting molecule in the chemical sensing deviceis an antigen that binds to an ovarian cancer marker antibody with anaffinity (K_(a)) of at least 10⁶ l/mole. In a more preferred embodimentthe K_(a) is at least 10⁸ l/mole. In another preferred embodiment theanalyte is a phospholipid. In a most preferred embodiment thephospholipid is lysophosphatidic acid (LPA).

In another embodiment, the invention provides an optical fiber, thefiber being shaped and adapted to provide a substrate surface for thechemical sensor. The fiber has a proximal end and a distal end. In oneembodiment, the fiber is shaped having a D-shape cross-section; inanother embodiment the distal end of the fiber is tapered to provide alarge substrate surface. In a more preferred embodiment the fiber has atleast two substrate surfaces.

In a yet additional embodiment, the invention provides an opticalcommunications device comprising a fiber, a plurality of particles, eachparticle comprising: a core, a shell having at least one surface andhaving contact with the core and wherein the shell comprises asulfur-oxygen molecular species, and wherein the particle has beenselectively sized using a notch filter and electromagnetic radiation,the electromagnetic radiation having a spectral wavelength of betweenabout 350 nm and about 1075 nm.

In a more preferred embodiment the optical communications devicecomprises a fiber, wherein the fiber is selected from the groupconsisting of ceramics, glasses, polymers, and metal-polymer composites.In one preferred embodiment the fiber cross-section is D-shaped. Inanother preferred embodiment the chemical sensor is disposed upon asurface of the fiber.

The invention also provides a method for synthesizing a chemical sensorcomprising gold nanoparticle aggregates, the method comprising the stepsof (i) providing one volume (1V) of a solution of 0.1 M HAuCl₄; (ii)diluting the solution of HAuCl₄ with Milli-Q water to a finalconcentration of between 4×10⁻⁴-6×10⁻⁴ M HAuCl₄; (iii) combining thediluted solution of HAuCl₄ with a 0.1 M solution of Na₂S to a finalconcentration of HAuCl₄ of between 4×10⁻⁵-6×10⁻⁵M HAuCl₄; (iv)incubating the combined solution for about between 60-120 minutes; and(v) measuring the extended plasmon band of the combined solution untilthe near-infra-red (NIR) absorption is at a wavelength longer than 600nm, thereby synthesizing a chemical sensing particle comprising goldnanoparticle aggregates. In one alternative, the 0.1 M solution of Na₂Sof step (iii) is substituted with an equal volume of 0.1M Na₂S₂O₃.

The invention also provides a method for coating gold nanoparticleaggregates onto a substrate, the method comprising the steps of (i)providing the gold nanoparticle aggregates as disclosed above; (ii)submerging the gold nanoparticle aggregates in a 5 mM aqueous solutionof a tethering molecule, the tethering molecule selected from the groupconsisting of trimethoxy[3-(methylamino)propyl]silane (APS) and(3-mercaptopropyl)trimethoxy silane (MPS), a compound having a silaneterminus and a thiol or amine terminus, and the like; (iii) incubatingthe gold nanoparticle aggregates with the tethering molecule to allowthe gold nanoparticle aggregates to adsorb the tethering molecules; (iv)providing a substrate, wherein the substrate is selected from the groupconsisting of silicon dioxide, silicon, and the like; (v) sonicating thesubstrate in contact with a 2% solution of surfactant, the surfactantselected from the group consisting of HELLMANEX, ALCONOX, a smallmolecule alkaline surfactant, and the like; (vi) sonicating thesubstrate with 18 mΩ water; (vii) drying the substrate under nitrogengas; (viii) depositing a volume of the adsorbed gold nanoparticleaggregates and tethering molecules solution onto the surface of thesubstrate; (ix) incubating the substrate with the solution for fiveseconds; (x) blowing the substrate dry with nitrogen gas; wherein theincubation time for step (iii) is selected from the group consisting of30 minutes, 60 minutes, 90 minutes, and 120 minutes, the methodresulting in gold nanoparticle aggregates coated onto a substrate.

The invention also provides a method for creating a chemical sensor withimproved sensitivity, the method comprising the steps of: (i) providingthe chemical sensing particle comprising gold nanoparticle aggregates asdisclosed above; (ii) illuminating the gold nanoparticle aggregates withan amplified femtosecond beam at a flux of approximately 0.1 mJ/cm² for1 hour; (iii) illuminating the gold nanoparticle aggregates using atunable picosecond laser, thereby creating a chemical sensor withimproved sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows representative electronic absorption spectra of goldnanoparticle aggregates in water during the growth and aggregationprocess.

FIG. 2 shows results from a persistent spectral hole burning experimentusing ˜800 nm laser light at 200 μJ/pulse.

FIG. 3 shows an illustration of how narrowing aggregate size/shapedistribution using light is achieved.

FIG. 4 shows a schematic illustration of how photoluminescence (PL) ofan SQD-Ab conjugate is quenched by an MNP-Ag conjugate.

FIG. 5 shows the results of two SDQ quenching experiments: two SERSspectra of an SQD-Ab (polyclonal donkey anti-goat Ab) conjugate havingbeen quenched by an MNP-Ag conjugate (pink and dark red) and one controlSERS spectrum of an SQD without a conjugated Ab (blue).

FIG. 6 is an illustration of how a Raman amplifier functions tointensify a photon beam.

FIG. 7A is an illustration of an exemplary optical fiber having asubstrate surface parallel to the axis of the fiber.

FIG. 8A illustrates an alternative exemplary optical fiber having asingle distal substrate surface at an angle to the axis of the fiber.

FIG. 9 illustrates the path of a photon within the fiber and showninteracting with a nanoparticle aggregate conjugated compound on thesubstrate surface of the fiber and the resulting SERS photon.

FIG. 10A illustrates another alternative exemplary optical fiber havingtwo distal substrate surfaces.

FIG. 11 illustrates the path of photons within the fiber and showninteracting with nanoparticle aggregate conjugated compound on thesubstrate surfaces of the fiber and the resulting SERS photons.

FIG. 12 illustrates the distal end of a fiber positioned in proximity tothe ends of two additional fibers that transmit the SERS photon to adetector.

FIG. 13 illustrates the angle of the distal substrate surface to thelongitudinal plane of the fiber.

FIG. 14 shows a representative UV-visible absorption spectrum of silvernanoparticles. The absorption towards the 780 nm region is believed tobe sufficient for SERS to occur.

FIG. 15 shows a Raman spectrum of bulk lysophosphatidic acid crystals(780 nm excitation, 3 mW power) for 16:0 LPA.

FIG. 16 shows a Raman spectrum of bulk lysophosphatidic acid crystals(780 nm excitation, 3 mW power) for 18:0 LPA.

FIG. 17 shows SERS spectra of one hundred samples of 10⁻⁶ M solutions of16:0 LPA and 18:0 LPA dried on silver nanoparticles (780 nm excitation,3 mW power).

FIG. 18 shows the SERS region between 1050 cm⁻¹-1150 cm⁻¹ for 100×10⁻⁶ Msolutions of 16:0 LPA and 18:0 LPA to show the distinguishable mode at1097 cm⁻¹ and 1101 cm⁻¹ (780 nm excitation, 3 mW power).

FIG. 19 illustrates a schematic of the Raman probe with a D-shaped (orside-polished) fiber coated with SERS substrate on the flat surface.Only the end segment of the fiber (about 1 cm) that is polished isshown. The rest of the unpolished fiber is about 0.5 m.

FIG. 20 illustrates the intensity distribution as light propagatesthrough the D-shaped fiber covered with a silver film. Area shown is across-section parallel to the polished surface and through the center ofthe fiber core (top view). Inset: contour plot of the intensitydistribution in a cross-section perpendicular to the fiber axis (endview). The semi-circle area is the fiber core with the cladding outsidethe half-circle. The thick black line above the semi-circle represents a0.1 mm metal particle thin film and above the film (the top bluerectangle) is the air.

FIG. 21 illustrates a representative SERS spectra of rhodamine 6G onsilver nanoparticles dried onto a D-shaped fiber collected withexcitation laser incident to fiber surface (A) and coupled into thefiber (B).

DETAILED DESCRIPTION OF THE INVENTION

The embodiments disclosed in this document are illustrative andexemplary and are not meant to limit the invention. Other embodimentscan be utilized and structural changes can be made without departingfrom the scope of the claims of the present invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a particle” includes aplurality of such particles, and a reference to “a surface” is areference to one or more surfaces and equivalents thereof, and so forth.

1: Unique Properties of Novel Gold Nanoparticle Aggregates (GNA)

We have recently discovered that the reaction of chloroauric acid(HAuCl₄) with sodium sulfide (Na₂S) results in generation of novel goldnanoparticle aggregates (GNAs). These GNAs have unique optical andsurface properties that are useful for applications, for example,surface enhanced Raman scattering (SERS), as a chemical sensor. SERS isa known and powerful technique for detecting molecules with highsensitivity and specificity. First, the GNAs we discovered are inaqueous solution and thereby naturally compatible with biologicalsamples in water. Second, the GNAs have strong near IR absorption(650-1200 nm) that is ideal for biological applications due to bettertissue penetration in this spectral region. Third, these GNAs haveunique surface properties due to sulfur species on their surface thatnot only causes the aggregation in the first place but also provides astrong driving force for binding with chemical and biological moleculeswith high affinity for sulfur. Some of the potential applications willbe outlined separately in the following sections.

The surface properties are undoubtedly due to the component sulfur andoxygen atoms (such as, but not limited to molecules of negativelycharged S_(x)O_(y), wherein x=1 or 2 and y=1, 2, 3, or 4).

The particles can have a size range of about between 60 and 200 nm.

FIG. 1 shows some representative electronic absorption spectra of theGNAs in water during the growth process. The spectrometer used limitsthe measurements to about 850 nm in the near IR. The strong near IR bandis clearly visible in the spectra.

2. Optical Control and Manipulation of Distribution of Size/Shape ofGold Nanoparticle Aggregates and its Application for Optical Filters

We have also discovered based on transient absorption and hole burningstudies that the broad near IR absorption of the GNAs is inhomogeneouslybroadened due to a distribution of sizes and/or shapes (see FIG. 2).Since the hole burned is permanent, this immediately led us to proposethe idea of using light (such as, but not limited to, high power lasers)to narrow the distributions of size/shape distribution. The idea isoutlined in FIG. 3 using a combination of “white light” and notchfilters to convert GNAs absorbing outside the notch filter coveredregion into GNAs that absorb only in the notch filter covered region. Asignificant increase in the number of GNAs in the notch filter coveredregion through this process is achieved.

This is useful since for many practical applications, for example, SERSand optical filters, it is highly desirable to narrow the distributionof size/shape of nanoparticle aggregates. For SERS in particular, theincident light has to be on resonance with the substrate absorption.Only those GNAs that have resonance absorption of the incident light areexpected to be SERS active. It is thus extremely beneficial to have anarrow size/shape distribution and thereby narrow optical absorption.

A white light source with a particular wavelength blocked by an opticalnotch filter is used to irradiate a sample of GNA having a broaddistribution of aggregate sizes and shapes. All the aggregates can beeither destroyed and/or converted into the aggregates that absorb at theblocked wavelength. This can significantly reduce the size and shapedistribution of the aggregates. Only aggregates that absorb (onresonance) with the blocked wavelength will remain and such aggregatesamples are stable. This principle can work for many other metalnanoparticle aggregates such as silver, Pt, and Pd, etc. Thesize/shape-narrowed aggregates are useful for many applications such asSERS or can be used as an optical filter with a fairly narrow bandwidth.The aggregates can further be patterned onto a solid substrate and usedfor any application that can benefit from narrow distributed metalnanoparticle aggregates.

FIG. 2 shows the results of a persistent spectral hole burningexperiment using ˜800 nm laser light at 200 μJ/pulse. Trace a) is priorto laser irradiation. Trace b) is after 2.5 hours of hole burning. Ahole is clearly seen in trace b) as well as a growth and shift of themaximum absorbance to bluer wavelengths. The SERS enhancement has beenobserved as being at about one-billion-fold (10⁹) that is approximatelythree to four orders of magnitude greater than that disclosed in theprior art. Such an increase in enhancement therefore results in areagent with greater sensitivity than other systems.

One potential application using this optical narrowing effect isproposed here. By exposing the aggregate solution to varying wavelengthsof light it may be possible to burn away all but a narrow absorptionband anywhere from 650 nm to 950 nm. This is shown schematically in theleft portion of FIG. 3. As shown in the right portion of FIG. 3, acomposition comprising particles having a narrow range of absorbance iscreated. By suspending these particles in glass, a low cost notch filteris produced. Current notch filters are highly expensive and difficult toproduce. With this technology it is possible to produce good qualitynotch filters at very low costs. While these filters might not be ashigh quality as current high cost filters, there is a large market forlow cost, low-end filters where high precision is not required. Themarket mainly comprises fiber-optical communications devices that areused to transmit information using photons instead of electrons. Thedevices comprise light amplifiers that retain the intensity of light asit travels through fiber optic networks. Light amplifiers are used toincrease the intensity of weak light signals as they travel through longdistances of fiber optic networks. The most commonly used form of lightamplification is the Raman amplifier because it has proven to be themost efficient. This technique increases the frequency of transmittedlight signals within a fiber optic network to prevent a loss oftransferred data. This market has not currently been tapped in notchfilters.

3. SERS Detection Applications for Sensing and Imaging

Raman spectroscopy is a powerful optical technique for detecting andanalyzing molecules. Its principle is based on detecting light scatteredoff a molecule that is shifted in energy with respect to the incidentlight. The shift, called Raman shift, is characteristic of individualmolecules, reflecting their vibrational frequencies that are like figureprints of molecules. As a result, the key advantage of Ramanspectroscopy is its molecular specificity while its main limitation isthe small signal due to low quantum yield of Raman scattering. One wayto enhance the Raman signal is to tune the excitation wavelength to beon resonance with an electronic transition, so called resonance Ramanscattering. This can usually produce an enhancement on the order of10²-10³. Another technique to enhance Raman scattering is surfaceenhancement by roughened metal surfaces, notably silver and gold, thatprovides an enhancement factor on the order of 10⁶-10⁸. Similar orsomewhat larger enhancement factors (˜10⁸-10¹⁰) have been observed formetal, mostly silver, nanoparticles.

In the last few years, it has been shown that an even larger enhancement(˜10¹⁰-10¹⁵) is possible for aggregates of metal nanoparticles, forexample, comprising silver and/or gold. The largest enhancement factorof 10¹⁴-10¹⁵ has been reported for rhodamine 6G (R6G) on single silvernanoparticle aggregates. This huge enhancement is thought to be mainlydue to significant enhancement of the local electromagnetic fields ofthe nanoparticle aggregates that absorb strongly the incident excitationlight for the Raman scattering process. With such large enhancement,many important molecules that are difficult to detect with Ramannormally can now be easily detected. This provides many interesting andnew opportunities for detecting and analyzing molecules using SERS withextremely high sensitivity and molecular specificity.

Of emphasis are the unique surface properties of the aggregates. It isthese surface properties that make the gold nanoparticles aggregate inthe first place and make the aggregates useful for SERS as a substratewith desirable properties.

Given the particular surface and optical features of the GNAs we havefound, they can be suitable for SERS detection and analysis of a largenumber of molecules, including, but not limited to, proteins, DNA,explosives, chemical and biological warfare agents, toxins, and evenvirus and biological cells. We have demonstrated that the GNAs are SERSactive for amino acids and DNA bases as well as antibodies for cancerdetection. As discussed in Section 2 above, the possibility of narrowingthe optical absorption of the GNAs for SERS is an important addedadvantage.

SERS can also be developed into a molecular imaging technique forbiomedical and other applications. Exciting Raman imaging equipment maybe usable for SERS imaging. SERS can provide an enhanced signal andthereby significantly shortened data acquisition time, making thetechnique practically useful for medical or other commercial andindustrial applications including, but not limited to, chip inspectionor chemical monitoring.

4. Antigen/Antibody Detection with Metal and SemiconductingNanoparticles

Fluorescent nanoparticles (semiconductor quantum dots, SQDs) have beenused recently as fluorescent biological markers and have been found tobe extremely effective. They offer advantages including higherstability, stronger fluorescence, tunability of color, and possibilityof optical encoding based on different sized or colored SQDs.

GNAs of the invention can be used to detect an analyte. Such an analytecan be, for example, but not limited to, an antigen, an antibody, abiochemical metabolite, an organic compound, a compound or elementhaving biological activity, or the like.

For example, the GNAs of the invention can be used in a novel dualoptical scheme for sensitive and selective detection of antigens and isillustrated in FIG. 4. The technique is based on detection ofphotoluminescence from SQDs with antibody (Ab) attached and SERS(surface enhanced Raman scattering) spectra of antigen (Ag) attached tothe GNAs that comprise metal nanoparticles (MNP). SERS spectrum is ameasure of the Raman spectrum of the Ag that can be significantlyenhanced by a metal nanoparticle. The Raman spectrum, similar to aninfra-red (IR) spectrum, is characteristic of specific molecules due tothe unique set of vibrational frequencies of each molecule. Before theAg and Ab interact or bind to one another, we expect strongphotoluminescence (PL) from the SQD and a well-defined SERS spectrum.Upon binding of Ab with the complementary Ag, two important consequencescan occur. First, the PL from the SQD will be significantly, if notcompletely, quenched by the GNA-Ab complex. Second, the SERS spectrum ofthe Ag will change with small but noticeable frequency shift and/orrelative spectral intensity changes due to Ag-Ab interaction. It is wellknown that PL from fluorophores can be significantly quenched whenbrought near a metal surface (bulk or nanostructured). The distance atwhich quenching occurs is a known parameter and is of the order ofbetween about 1-2 nm for effective quenching. The distance will bedependent on the sizes of the SQD, GNA, Ag, and Ab, the length of alinker molecule (LM) between the SQD and the Ag (or alternatively,between the SQD and the Ab, between the GNA and Ab, or between the GNAand Ag), as well as on their relative binding configuration. We canestimate at this point that a certain favorable configuration and sizewill allow quenching. This is supported by a recent report from Wang etal. that PL quenching of a small, green-emitting QD with antibodyattached by large, red-emitting QD with antigen attached occurs when theAg and Ab interact (Wang et al. (2002) NanoLett., 2: 817-822). Thisexperiment demonstrates that the distance can be close enough foreffective PL quenching due to resonance energy transfer from the largeQD to the smaller QD.

As for the SERS aspect, we have very recently demonstrated that we candetect the SERS spectrum of a polyclonal Ab attached to goldnanoparticles through electrostatic interaction. FIG. 5 shows tworepresentative SERS spectra where the reproducible peaks can beattributed to the Ab. To our best knowledge, this is the firstdemonstration of SERS detection of Ab. Since Raman or SERS spectrum isextremely sensitive to the structure, configuration and environment of amolecule, we anticipate that the SERS spectrum of an Ab can change uponinteraction with its Ag.

The proposed scheme offers a novel technique for detecting antigen withhigh sensitivity (offered by both PL and SERS) and specificity (offeredby both SERS and Ag-Ab interaction). In a typical experiment, we choosea laser wavelength that is on resonance with absorption of the QD(usually in the near UV and visible region) for measuring PL from theSQD with Ab attached, with and without binding to Ag attached to GNA. Wethen choose a laser wavelength that is off-resonance for the SQDabsorption and produces no PL for SERS measurement (usually near IR) ofthe Ag attached to GNA, with and without binding with Ab attached toSQD. Comparing the two situations with and without Ag-Ab binding, we cansee PL quenching of the SQD and SERS spectral change upon binding. Sincethe PL quenching of the SQD by a GNA is expected to be much moreeffective than a large SQD, the PL can be completely quenched in thesituation with Ag-Ab binding. This is thus a zero-background experimentand can be much more sensitive than conventional PL detection that istypically not zero-background. For SERS, since the enhancement can be ashigh as 10⁹, it is almost as sensitive as fluorescence but has theextremely important advantage of direct molecular specificity betweenthe Ag and the Ab.

In another example, the Ag can be replaced by a second Ab, the second Abbeing specific for binding the first Ab. The second antibody can be fromthe same animal species as the first Ab, or can be from another animalspecies. Such first and second Abs are well known to those in the artand can be raised in and isolated from an animal such as, but notlimited to, a rabbit, a human, a mouse, a rat, a monkey, an ape, a goat,a sheep, a cow, a pig, a donkey, a horse, a guinea pig, a whale, awombat, a platypus, or the like.

SERS is also useful for detecting other cancer biomarkers that caninteract or bind to the GNA surface. For example, Sutphen et al. haverecently shown that lysophospholipids (LPL) are potential biomarkers ofovarian cancer (Sutphen et al. (2004) Cancer Epidemiol Biomarker Prev.,13: 1185-1191). Based on the molecular structure of LPL molecules, afavorable interaction between LPL molecules with GNA throughelectrostatic interaction can occur at the appropriate pH. In the caseof the SERS experiment using a polyclonal Ab shown in FIG. 5, thestrongest interaction with GNA occurs at the isoelectrostatic pH, i.e.pH at which the GNA has equal number of positive and negative charges.The pH is varied to adjust the charge on the GNA to determine theoptimal pH or charge for strong interaction with LPL.

By conjugating fluorescent nanoparticle QDs to antigens and mixing theAg-QD conjugate with a GNA-Ab composition, quenching of fluorescenceupon binding of the antigen/antibody pair can be observed. The Ag and/orthe Ab can be conjugated to the QD or GNA using a linker molecule (LM).A decrease in fluorescence can indicate the presence of the antibody forthat particular antigen to which the fluorescing QDs have been attached.Depending on which antigen is utilized a wide array of antibodies can bedetected. This can allow for the rapid detection of cancers or diseasesthat currently can take days or weeks to diagnose. Likewise, the schemecan work as well if antibody is attached to a fluorescent QD and therespective antigen to a metal nanoparticle. Metal particles have noflorescence with visible excitation. The fluorescence quenching by metalnanoparticles can be more effective than quenching by larger QDs. Thisapproach is sensitive and specific. The distance between the metalnanoparticle and QD is important for this to work (for example, thedistance can be less than 2 nm). The interaction between the twocomponents can be adjusted to achieve the maximum quenching effect.

5. Detection of Tumor Markers

Surface-enhanced Raman scattering using silver nanoparticles was appliedto detect various forms of lysophosphatidic acid (LPA) to examine itspotential application as an alternative to current detection methods ofLPA as biomarkers of ovarian cancer. Enhancement of the Raman modes ofthe molecule, especially those related to the acyl chain within the800-1300 cm⁻¹ region, was observed. In particular, the C—C vibrationmode of the gauche-bonded chain around 1100 cm⁻¹ was enhanced to allowthe discrimination of two similar LPA molecules. Given the molecularselectivity of this technique, the detection of LPA using SERS mayeliminate the need for partial purification of samples prior to analysisin cancer screening.

Lysophosphatidic acid (LPA), originally known for its role as anintermediate in intracellular lipid metabolism, has now been recognizedas an important multifunctional biological mediator that can elicitcellular responses including mitogenic and antimitogenic effects on thecell cycle, actin skeleton regulation, and cellular motility (see Tigyiet al. (1994) Proc. Nat. Acad. Sci. 91: 1908-1912; van Corven et al.(1989) Cell 59: 45-54; Ridley and Hall (1992) Cell 70: 389-399; and Zhouet al (1995) J. Biol. Chem. 270: 25549-25556). The involvement of LPA ininducing cell proliferation, migration and survival implicates it in theinitiation and progression of malignant disease, and has been proposedas a sensitive biomarker for ovarian cancer (see Xu et al (1998) JAMA280: 719-723; Mills and Moolenaar (2003) Nature Reviews 3: 582-591; Fanget al (2004) J. Biol. Chem. 279: 9653-9661; and Sutphen et al (2004)Cancer Epidemiol. Biomark. Prev. 13: 1185-1191).

Typically, the detection of LPA has been conducted using chromatographyand mass spectroscopy assays that require a partial purification of thesamples using thin layer chromatography (TLC) prior to analysis.Although this method is effective, an underestimation of LPAconcentration can result during the recovery process due in part to thevarying mobility of the LPA salts (free acid, sodium and calcium salts)when subjected to chromatography by TLC. The low stability of LPA alsocalls for fast and sensitive detection techniques.

A powerful optical detection technique based on surface-enhanced Ramanscattering (SERS) offers a unique combination of high sensitivity andmolecular specificity. With SERS, the Raman signal of a molecule isincreased by many orders of magnitude as a result of strong enhancementof the excitation light through the resonance of the metal's surfaceelectrons called the surface plasmon (see Moskovitz (1985) Rev. ModernPhysics 57: 783-828; Otto et al. (1992) J. Phys. Condense Matter 4:1143-1212; and Campion and Kambhampati (1998) Chem. Soc. Rev. 27:241-250). SERS has been successfully used in the detection and analysisof a large number of chemicals and biological molecules (see Albrechtand Creighton (1977) J. Am. Chem. Soc. 99: 5215-5217; Nie and Emory(1997) Science 275: 1102-1106; Keating et al. (1998) J. Phys. Chem. B102: 9414-9425; Kneipp et al (1998) Phys. Rev. E 57: R6281-R6284; andSchwartzberg et al. (2004) J. Phys. Chem. B 108: 19191-19197).

6. SERS Application for Detection and Analysis of SemiconductorNanoparticles

Another application of SERS based on the gold nanoparticle system is formeasuring Raman spectrum of semiconductor nanoparticles (QDs). Similarto molecules, normal Raman signals are very small and thus Ramanspectrum is challenging to measure. SERS as an enhanced Raman techniquefor measuring Raman for semiconductor nanoparticles have not beenreported before. The surface chemistry of the metal nanoparticles andthe semiconductor QDs must be compatible for this to work. The sulfurspecies on the surface of the GNAs are ideal for II-VI SQDs to bind,enabling SERS detection of the SQDs. This provides a powerful method fordetecting and analyzing semiconductor nanoparticles.

7. SERS for Raman Amplifier in Optical Communications

Raman amplifiers have been used to amplify signal in opticalcommunications (see, for example, FIG. 6). SERS can provide moreamplification than normal Raman amplifiers. By doping MNPs, for exampleGNAs, into glass or polymer fibers, Raman scattering from the glass orpolymer matrix can be used to amplify optical signal with the properwavelength.

8. Synthesis of Biological Molecules Chemical Synthesis of Peptides

Proteins or portions thereof may be produced not only by recombinantmethods, but also by using chemical methods well known in the art. Solidphase peptide synthesis may be carried out in a batchwise or continuousflow process which sequentially adds α-amino- and side chain-protectedamino acid residues to an insoluble polymeric support via a linkermolecule. A linker molecule such as methylamine-derivatized polyethyleneglycol is attached to poly(styrene-co-divinylbenzene) to form thesupport resin. The amino acid residues are N-α-protected by acid labileBoc (t-butyloxycarbonyl) or base-labile Fmoc(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected aminoacid is coupled to the amine of the linker group to anchor the residueto the solid phase support resin.

Trifluoroacetic acid or piperidine are used to remove the protectinggroup in the case of Boc or Fmoc, respectively. Each additional aminoacid is added to the anchored residue using a coupling agent orpre-activated amino acid derivative, and the resin is washed. Thefull-length peptide is synthesized by sequential deprotection, couplingof derivatized amino acids, and washing with dichloromethane and/orN,N-dimethylformamide. The peptide is cleaved between the peptidecarboxy terminus and the linker group to yield a peptide acid or amide.These processes are described in the Novabiochem 1997/98 Catalog andPeptide Synthesis Handbook (San Diego Calif. pp. S1-S20). Automatedsynthesis may also be carried out on machines such as the ABI 431Apeptide synthesizer (ABI). A protein or portion thereof may be purifiedby preparative high performance liquid chromatography and itscomposition confirmed by amino acid analysis or by sequencing (Creighton(1984) Proteins, Structures and Molecular Properties, W H Freeman, NewYork N.Y.).

In particular, a purified antigen may be used to produce antibodies orto screen libraries of pharmaceutical agents to identify those thatspecifically bind an antigen. Antibodies to an antigen may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of polyclonal antibodies, various hosts includinggoats, rabbits, rats, mice, humans, and others may be immunized byinjection with an antigen or with any fragment or oligopeptide thereofthat has immunogenic properties. Rats and mice are preferred hosts fordownstream applications involving monoclonal antibody production.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface-activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol. Amongadjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable. (For review of methodsfor antibody production and analysis, see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.)

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to an antigen have an amino acid sequence consistingof at least about 5 amino acids, and, more preferably, of at least about14 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofantigen amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

Antibodies

Monoclonal antibodies to an antigen may be prepared using any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, for example, Kohler et al. (1975) Nature256: 495-497; Kozbor et al. (1985) J. Immunol. Methods 81: 31-42; Coteet al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; and Cole et al.(1984) Mol. Cell Biol. 62: 109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, for example, Morrison et al.(1984) Proc. Natl. Acad. Sci. 81: 6851-6855; Neuberger et al. (1984)Nature 312: 604-608; and Takeda et al. (1985) Nature 314: 452-454.)Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceantigen-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries.(See, for example, Burton (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, for example, Orlandi et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter et al. (1991) Nature 349: 293-299.)

Antibody fragments that contain specific binding sites for an antigenmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, forexample, Huse et al. (1989) Science 246: 1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity and minimal cross-reactivity. Numerousprotocols for competitive binding or immunoradiometric assays usingeither polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the measurement of complex formation between an antigen and itsspecific antibody. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering antigen epitopesis preferred, but a competitive binding assay may also be employed.(Maddox, supra.)

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for an antigen. Affinity is expressed as an associationconstant, K_(a), which is defined as the molar concentration ofantigen-antibody complex divided by the molar concentrations of freeantigen and free antibody under equilibrium conditions. The K_(a)determined for a preparation of polyclonal antibodies, which areheterogeneous in their affinities for multiple antigen epitopes,represents the average affinity, or avidity, of the antibodies for anantigen. The K_(a) determined for a preparation of monoclonalantibodies, which are monospecific for a particular antigen epitope,represents a true measure of affinity. High-affinity antibodypreparations with K_(a) ranging from about 10⁹ to 10¹² l/mole arepreferred for use in immunoassays in which the antigen-antibody complexmust withstand rigorous manipulations. Low-affinity antibodypreparations with K_(a) ranging from about 10⁶ to 10⁷ l/mole arepreferred for use in immunopurification and similar procedures whichultimately require dissociation of antigen, preferably in active form,from the antibody. (See Catty (1988) Antibodies, Volume I: A PracticalApproach, IRL Press, Washington, D. C.; and Liddell and Cryer (1991) APractical Guide to Monoclonal Antibodies, John Wiley & Sons, New York,N.Y.)

The titre and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody.ml⁻¹,preferably 5-10 mg specific antibody.ml⁻¹, is preferred for use inprocedures requiring precipitation of antigen-antibody complexes.Procedures for evaluating antibody specificity, titer, and avidity, andguidelines for antibody quality and usage in various applications, aregenerally available. (See, for example, Catty, supra, and Coligan et al.supra.)

Preparation and Screening of Antibodies

Various hosts including, but not limited to, goats, rabbits, rats, mice,and human cell lines may be immunized by injection with antigen or anyportion thereof. Adjuvants such as Freund's, mineral gels, andsurface-active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, KLH, and dinitrophenol may be usedto increase immunological response. The oligopeptide, peptide, orportion of protein used to induce antibodies should consist of at leastabout five amino acids, more preferably ten amino acids, which areidentical to a portion of the natural protein. Oligopeptides may befused with proteins such as KLH in order to produce antibodies to thechimeric molecule.

Monoclonal antibodies may be prepared using any technique that providesfor the production of antibodies by continuous cell lines in culture.These include, but are not limited to, the hybridoma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique. (See,for example, Kohler et al. (1975) Nature 256:495-497; Kozbor et al.(1985) J. Immunol. Methods 81:31-42; Cote et al. (1983) Proc. Natl.Acad. Sci. 80:2026-2030; and Cole et al. (1984) Mol. Cell. Biol. 62:109-120.)

Alternatively, techniques described for antibody production may beadapted, using methods known in the art, to produce epitope-specific,single chain antibodies. Antibody fragments that contain specificbinding sites for epitopes of the protein may also be generated. Forexample, such fragments include, but are not limited to, F(ab′)2fragments produced by pepsin digestion of the antibody molecule and Fabfragments generated by reducing the disulfide bridges of the F(ab)2fragments. Alternatively, Fab expression libraries may be constructed toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity. (See, for example, Huse et al. (1989) Science 246:1275-1281.)

The antigen, or a portion thereof, may be used in screening assays ofphagemid or B-lymphocyte immunoglobulin libraries to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoassays using either polyclonal or monoclonal antibodieswith established specificities are well known in the art. Suchimmunoassays typically involve the measurement of complex formationbetween the protein and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes is preferred, but a competitive bindingassay may also be employed (Pound (1998) Immunochemical Protocols,Humana Press, Totowa N.J.).

In the alternative, an antibody can be substituted by, for example, achimeric protein that comprises a portion or fragment of a T-cellreceptor (TCR). TCRs have an immunoglobulin domain that binds acell-surface antigen comprising a host or a non-host molecule. Suchmolecules can be of viral origin or can be a particular cancer markerprotein. The chimeric protein can also comprise a soluble protein (i.e.present in a bodily fluid or the cell cytoplasm) or a cellmembrane-associated protein (such as a ligand receptor, an ion channel,or a molecule involved in signal transduction.

Metal nanoparticles are currently studied for a wide variety ofbiomedical applications including contrast imaging, ultrasonic imaging,thermal destruction of specific cancer cells, and laser tissue welding.All applications of this type rely on the optical and physicalproperties associated with metal nanoparticles, nominally of gold. Muchof this work has focused on gold nanoshells due to their near IR opticalabsorption where tissue transmission is at its peak, making in-vivoapplications feasible. This gold nanoparticle aggregate system possessesthese same optical features with multiple advantages. While nanoshellscan be tuned to absorb in a particular region, their absorption isinhomogenously broad and cannot be narrowed without significantpurification. Therefore a significant percentage of nanoshells will befunctionally useless at a given wavelength. Gold aggregates on the otherhand can be tuned to have a very narrow absorption through the opticalhole burning technique. With the absorption tuned to a given wavelengthall aggregates will be utilized making them significantly more efficientfor any of the above applications.

One of the most exciting of these applications is thermal destruction ofcancer cells. The nanoparticle aggregates are selectively attached tocancer cells in a tumor by a passive mechanism that has been termed an“enhanced permeability and retention effect”. The tumor mass is thenilluminated with near IR laser light which passes harmlessly through thetissue, but is absorbed strongly by the aggregates, causing them to heatdrastically, killing only the cancerous cells. (See O'Neal et al. (2004)Cancer Lett. 209: 171-176, herein incorporated by reference in itsentirety.) This technology has been utilized with gold-silica nanoshellsfurther comprising “stealthing” polymers, such as poly(ethyleneglycol)and derives thereof, or liposomes; however this can be done better withgold nanoparticle aggregates of the present invention.

9. Detection of Specific Compounds Using Optical Fibers

The nanoparticle aggregates can be used to detect specific compoundsthat may be at very low levels in a sample. Such a sample can be blood,urine, saliva, lung lavage, gastric fluid, lymphatic fluid, any otherbody fluid, or the like. In addition, the sample can be a sample ofwater or other aqueous medium, such as water from a spring, a stream, ariver, a pond, a lake, a sea, or an ocean. The sample can be ageological sample such as from a geothermal spring, a lava evaporate orexudate, a hydrocarbon, or from an abyssal trench; a plant sample suchas from the xylem or phloem of a stalk or trunk; a sample from a fluidin a man-made structure such as concrete, cement, aggregate, or thelike; a sample of fluid from a piece of machinery such as an engine,motor, compressor, or the like.

The nanoparticle aggregates can be conjugated with antibody, theantibody having been synthesized to bind a specific compound. Such aspecific compound can be a protein, a fatty acid, a carbohydrate, anorganic compound based upon a benzene ring structure, an organiccompound based upon a short chain hydrocarbon, a medium chainhydrocarbon or a long chain hydrocarbon. The specific compound can bemodified with a reactive group. Such reactive groups are well known tothose of skill in the art and can include phosphate groups, methylgroups, hydroxyl groups, sulphate groups, acetyl groups, or the like.

The nanoparticle aggregate-antibody conjugate complex has an alteredSERS profile when the specific compound binds to the complex. Thecomplex can be applied and incorporated onto a substrate surface of anoptical fiber as disclosed herein. The exterior surface of the fibercomprises a compound that reflects photons. Such compounds are wellknown to those in the fiberoptic arts. The optical fiber has a proximalend or proximal section and a distal end or distal section. The distalend of a conventional fiber has a circular cross-section. A modifiedsubstrate surface can be created to create a substrate surface having anarea larger than that of the distal end. The substrate surface of thefiber is created by removing a distal portion of the fiber sectionthereby creating a fiber with a distal end cross-section that isdifferent from the distal end of the original fiber. The cross-sectioncan be D-shaped, diamond-shaped, triangular, oval, or anothernon-symmetrical shape. Removing a portion of the fiber results insubstrate surface with a larger surface area than the surface area ofthe original end of the fiber.

The resulting substrate surface can have a surface area that is up to atleast about 8,000-fold larger than the distal end surface of theoriginal fiber. The diameter of the fiber can be from between about 0.01μm to about 10 μm. In one alternative, the diameter is from betweenabout 0.1 μm to about 1 μm. In another alternative, the diameter isbetween about 0.2 μm to about 8 μm.

The complex is applied and incorporated onto the substrate surface andlight is directed longitudinally through the fiber. The light can becoherent and/or non-coherent. The light interacts with the nanoparticleaggregate-antibody conjugate complex and a resulting SERS profile can becompared with a SERS profile from the nanoparticle aggregate-antibodyconjugate complex that is bound with a known amount of specificcompound. The SERS radiation is detected using a photon detectorsuitably disposed to detect the SERS radiation. The detector can bedisposed at or near the substrate surface of the fiber at the distal endor distal section of the fiber, at or near the proximal end or proximalsection of the fiber, or at another position as disclosed herein.

The fiber can have one or more such substrate surfaces. In the case oftwo substrate surfaces, the second substrate surface can reflect theSERS signal from the first substrate surface to the detectorlongitudinally along the length of the fiber, resulting in a markedlyimproved amplification of the SERS signal. Similarly, the firstsubstrate surface can reflect a SERS signal from the second substratesurface to the detector.

In another alternative, at least one additional optical fiber can bepositioned in proximity to the distal end or distal section of thefiber. The end of the additional fiber can have the same shape as theshape of the distal end or distal section of the fiber, such that SERSradiation emitted from the fiber is conducted through the additionalfiber to a detector. Two additional fibers can be used in parallel wherethere are two new substrate surface s on the fiber.

The optical fiber can additionally have a non-uniform diameter, forexample, the distal end having a cross-section perpendicular to thelongitudinal plane that is larger in magnitude than a cross-section ofthe proximal end. Such a shape can further increase the amount of SERSradiation produced by a photon source.

The optical fiber can be made using glass, ceramics, or the like; or apolymeric compound such as cyclic olefin polymer (COP), polysulfone (forexample, UDEL and RADEL resins), fluorinated terpolymers (such as thosesynthesized from tertafluoroethylene, hexafluoropropylene, andvinylidene fluoride), polycarbonate, polyacrylate, polystryrene, or thelike.

As illustrated in FIG. 13, the surface of the substrate surface (2) ofthe fiber (1) is at an angle θ (7) to the longitudinal plane (8) of thefiber. The angle θ is between 15° and 75°; preferably between 15° and65°; more preferably between 15° and 45°; and most preferably between15° and 35°. In one example, the angle θ is 22.5°. The optimalpreferable angle can be determined empirically to find the optimal anglefor back-scattering of the SERS radiation.

One of skill in the art can readily determine which angle θ is optimalusing data collected from experimentation as described above andknowledge of the composition of the fiber. It is well known in the artthat different compositions have different refractive indices and one ofskill in that art would know that different compositions will have aparticular optimal refractive index. One of skill in the art would alsoknow that it is not always necessary to create a fiber having a distalend section having a substrate surface at the optimal angle θ to theplane of the fiber since the nanoparticle aggregate-antibody conjugatecomplex can have different effects upon the refractivity of a fibercompound. It would require relatively little experimentation by one ofskill in the art to determine the optimal preferable angle θ.

The SERS radiation can be further enhanced approximately 4-5-fold if anelectrical field of a few Volts per centimeter (V/cm) is applied acrossthe fiber, approximately perpendicular to the substrate surface. Thepotential difference can be maintained through an electricallyconducting solution. The electrically conducting solution can be aqueousor non-aqueous but should not quench SERS radiation to the extent thatthe SERS enhancement due to the electrical field is quenched by theelectrically conducting solution.

Examples of different optical fiber ends are illustrated in FIGS. 7through 12. FIG. 7A illustrates a cylindrical fiber (1) having anexterior surface (3) and having a longitudinal portion removed from thedistal section of the fiber thereby creating a substrate surface (2).FIG. 7B shows a cross-section of the distal section.

FIG. 8A illustrates an alternative distal section whereby a longitudinalportion is removed from the fiber at an included plane to the fiberresulting in a tapered distal end of the fiber. FIGS. 8B, 8C, and 8Dillustrate three cross-sections of the distal section at differentpositions along the length of the fiber. FIG. 9 illustrates the path ofa photon (4) from a photon source and the path of a SERS photon (5) fromthe substrate surface of the fiber to a detector (6).

FIG. 10A illustrates another alternative distal section whereby twolongitudinal portions are removed from the fiber resulting in a tapereddistal end of the fiber. FIGS. 10B, 10C, and 10D illustrate threecross-sections of the distal section at different positions along thelength of the fiber. FIG. 11 illustrates the path of a photon (4) fromthe photon source and the path of a SERS photon (5) from the substratesurface (2) of the fiber to a detector (6).

FIG. 12 illustrates the distal end of a fiber positioned in proximity tothe ends of two additional fibers that transmit the SERS photon to adetector. FIG. 13 illustrates the angle between the substrate surface(2) and the longitudinal plane (8) or axis of the fiber.

The nanoparticle aggregates can be formed and shaped into a desiredshape, such as a sphere, a cylinder, a rod, a cone, a pyramid, or othershape, not limited to regular shapes, and deposited upon a substrate ata desired density using means well known t to hose of skill in the art.(See, for example, Fan et al. (2005) J. Vac. Sci. Technol. 8: 947-953;Chaney et al. (2005) Appl. Phys. Lett. 87: pub. no. 031908.)

EXAMPLES

The invention will be more readily understood by reference to thefollowing examples, which are included merely for purposes ofillustration of certain aspects and embodiments of the present inventionand not as limitations.

Example I Synthesis of Gold Nanoparticle Aggregates

The synthesis of the gold nanoparticle aggregates (GNAs) was performedas follows: 400-600 ul of a 0.02 M HAuCl₄ stock solution was diluted to4×10⁻⁴-6×10⁻⁴ M with Milli-Q water in glassware cleaned in aquaregia andrinsed with Milli-Q water to avoid contamination. To this, 40-60 μl of a0.1 M solution of Na₂S that has been aged for 2-3 months was added.After approximately 60-120 minutes, the color changed from a strawyellow to deep purple with the extended plasmon band (EPB) growing inbetween 600-1000 nm, indicating reaction completion. The aggregateformation was signified by strong near-infra-red (NIR) absorption atwavelengths longer than 600 nm. This reaction is also performed withsodium thiosulfate (Na₂S₂O₃) by replacing 1:1 the sodium sulfidesolution, however, while these particles are optically identical tothose that are sodium sulfide generated, they work poorly for SERS andshould only be used for non detection applications. HAuCl₄, Na₂S,Na₂S₂O₃ and were obtained from Sigma-Aldrich (St. Louis Mo.) at thehighest level of purity available.

Example II Coating Gold Nanoparticle Aggregates onto Substrates/Fiber

The coating of gold nanoparticle aggregates onto a substrate/fiber wasperformed in two ways. First, by spin coating or drop casting a dilutesolution of the aggregates onto the substrate. This yields a relativelythick film, however, it is not as stable as is necessary for manyapplications. The second method utilizes a tethering molecule, in thiscase trimethoxy[3-(methylamino)propyl]silane (APS). The substrate wascleaned prior to the silanization step by sonication in 2% solution ofHELLMANEX or other surfactant, followed by 18 mΩ water.

The gold nanoparticle aggregates were then submerged in a 5 mM aqueoussolution of APS to deposit the tethering molecules. After 1-2 minutesthe substrate was rinsed with water, dried under nitrogen, and 40 μl ofthe aggregate solution was placed on the surface. After several secondsexposed to the solution, it was blown dry with nitrogen. This provided asignificantly thinner film than those made by spin or drop casting,however, it was extremely stable and robust under use. This film wasmade to near monolayer coverage by increasing the substrate exposuretime to the APS and aggregate solutions to approximately 1 hour. APS wasobtained from Sigma-Aldrich.

Example III Band Narrowing in Gold Nanoparticle Aggregates

The extended plasmon band of the gold nanoparticle aggregates were tunedand narrowed via a regeneratively amplified, mode locked femtosecondTi-sapphire laser system. This was done by illuminating the sample withthe amplified femtosecond beam at a flux of approximately 0.1 mJ/cm².After approximately 1 hour, a deep, broad hole was burned in theabsorption spectrum at near 800 nm while absorption to the bluedrastically increased.

In order to tune the band to one particular wavelength a tunablepicosecond laser is required. Due to the broad spectral linewidth of thefemtosecond pulses it is impossible to completely narrow the extendedplasmon band (EPB). By using the spectrally narrow picosecond pulses itis possible to selectively destroy the EPB to the blue and red of thedesired excitation wavelength, enhancing the absorption more than twofold.

Example IV Single Particle SERS/Luminescence and Bulk SERS

Samples for single particle experiments were prepared by immobilizingthe GNAs or semiconductor quantum dots (SQD) on glass coverslips withtrimethoxy[3-(methylamino)propyl]silane (APS). Coverslips were cleanedprior to the silanization step by sonication in 2% solution ofHellmanex, followed by 18 MΩ water. They were then submerged in 5 mMaqueous solution of APS to deposit the tethering molecules. After 1-2minutes the coverslips were rinsed with water, dried under nitrogen, and40 μl of the aggregate or SQD solution was placed on one surface. Afterseveral seconds exposed to the solution, it was blown dry with nitrogen.

Single particle experiments were performed on a custom designed confocalmicroscope built onto an inverted fluorescence microscope (Axiovert 100,Carl Zeiss, Inc., Thornwood N.Y.). A helium-neon or Argon ion laserdepending on desired excitation wavelength was coupled into the backport of the microscope and directed into a high numerical apertureobjective (Apochromat 100×, 1.4 NA) that focused the light onto thesample surface. The sample was then raster scanned across the focusedlaser to generate an image using a commercially available piezoelectricscanner (Physik Instrumente, Auburn Mass.) and control electronics(Digital Instruments (Veeco Instruments Inc.) Woodbury N.Y.). The Ramanscattered light or fluorescence was collected with the same objectiveused for excitation and focused onto a confocal aperture. The Rayleighscattered light was then removed using a holographic notch filter(Kaiser Optical Systems, Inc., Ann Arbor Mich.) and the remainingscattered light was focused onto an avalanche photodiode (EG&G (ClTYSTATE)). Once a nanoparticle aggregate or QD was located, it wascentered on the focused laser and the Raman scattering was directed intoa spectrograph (Acton Instruments, Acton Mass.) that dispersed the lightonto a liquid nitrogen cooled CCD camera (Princeton Instruments, TrentonN.J.). Typically six spectra (30 seconds each) were averaged. Bulk SERSexperiments were performed on a Renishaw MICRORAMAN instrument (RenishawPlc, Wotton-under-Edge, GL12 8JR, United Kingdom) with a 783 nm diodeexcitation laser. A drop of sample was placed on a quartz substrate andthe laser was focused into the solution. Typically 4 spectra (30seconds) were averaged.

Example V Surface-Enhanced Raman Scattering Detection ofLysophosphatidic Acid

Lysophosphatidic acid (LPA), originally known for its role as anintermediate in intracellular lipid metabolism, has now been recognizedas an important multifunctional biological mediator that can elicitcellular responses including mitogenic and antimitogenic effects on thecell cycle, actin skeleton regulation, and cellular motility. Theinvolvement of LPA in inducing cell proliferation, migration andsurvival implicates it in the initiation and progression of malignantdisease, and has been proposed as a sensitive biomarker for ovariancancer.

Typically, the detection of LPA has been conducted using chromatographyand mass spectroscopy assays that require a partial purification of thesamples using thin layer chromatography (TLC) prior to analysis.Although this method is effective, an underestimation of LPA levels canresult during the recovery process due in part to the varying mobilityof the LPA salts (free acid, sodium and calcium salts) when subjected tochromatography by TLC. The low stability of LPA also calls for fast andsensitive detection techniques.

A powerful optical detection technique based on surface-enhanced Ramanscattering (SERS) offers a unique combination of high sensitivity andmolecular specificity. With SERS, the Raman signal of a molecule isincreased by many orders of magnitude as a result of strong enhancementof the excitation light through the resonance of the metal's surfaceelectrons called the surface plasmon. SERS has been successfully used inthe detection and analysis of a large number of chemicals and biologicalmolecules.

Here we report for the first time to our knowledge, the application ofSERS using silver nanoparticles as a potential alternative technique fordetecting LPA with high sensitivity and molecular specificity.Experimental results obtained for 16:0 LPA and 18:0 LPA successfullydemonstrated not only that SERS of LPA can be measured but also that theSERS spectra of the two very similar LPA molecules were shifted enoughin the 1100 cm⁻¹ region to uniquely identify them. The results suggestedthe strong potential for practical LPA detection using SERS-basedtechniques that are fast, sensitive, and molecular specific.

Powder samples of 16:0 LPA(1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphate(sodium salt)) and 18:0 LPA(1-Stearoyl-2-hydroxy-sn-glycero-3-phosphate(sodium salt)) werepurchased from Avanti Polar Lipids, Inc. Silver nitrate and sodiumcitrate were purchased from Sigma Aldrich (St Louis Mo.). Raman spectrawere obtained using a Renishaw micro-Raman setup with a 50× objectivelens and 780 nm excitation laser at 3 mW.

Silver nanoparticles were prepared using a synthesis from Lee and Meiselusing silver nitrate as the metal precursor and a sodium citratereducing agent (Lee and Meisel (1982) J. Phys. Chem. 86: 3391-3395).Formation of the silver nanoparticles was monitored using UV-visiblespectroscopy using a HP 8452A spectrometer with 2 nm resolution. Thisnanoparticle solution was then concentrated by a factor of 10 viacentrifugation prior to application. For the SERS experiment, 2 μl dropsof the concentrated silver nanoparticles were placed on a glass slideand allowed to dry. Its Raman signal was obtained with one accumulationof a 30 second scan. After the silver had dried, 4 μl of a 100 μMsolution of either the 16:0 LPA or 18:0 LPA (dissolved in Milli-Q water)was added on top of the silver to dry. The 4 μl volume ensured thecomplete coverage of the silver that had dried on the glass. The Ramansignal was then collected using the same scan parameters. Forcomparison, Raman of the crystalline LPA samples was collected with fiveaccumulations of a two-minute scan.

The primary goal of this work was to demonstrate the ability of SERS tobe selective, reproducible, and sensitive in detecting 16:0 LPA versus18:0 LPA and show its potential as a viable alternative to currentdetection methods. FIG. 14 presents the UV-vis absorption spectrum ofthe silver nanoparticles used in this experiment. The surface plasmonband of these particles peaks near 400 nm, and contains very weakabsorption around 780 nm, possibly due to some nanoparticle aggregation.For SERS to work effectively, resonance absorption of the metalnanoparticle substrate with the incident wavelength is essential(Schwartzberg et al. (2004) supra). The fact that there is only weakabsorption near 780 nm while SERS has been demonstrated to workeffectively suggests that either the weak absorption at 780 nm is enoughfor SERS occur, or the nanoparticles have further aggregated uponconcentrating and drying for the SERS experiment that may have caused ared-shift of the absorption band and increased absorption at 780 nmsimilar to what has been observed for gold nanoparticles (Norman et al.(2002) J. Phys. Chem. B 106: 7005-7012).

The Raman spectra and molecular structures of the bulk crystal of 16:0LPA and 18:0 LPA are presented in FIG. 15 and FIG. 16, respectively.With the only difference between the two LPAs being the length of theiracyl chains, the ability to apply SERS for detection applicationsdepends on its capacity to detect the acyl peaks. Hence, experimentalmeasurements were performed between 800-1400 cm⁻¹ where many of the acylpeaks occur. The vibrational modes were assigned based on characteristicRaman frequencies (Lin-Vien et al. (1991) In: Infrared and RamanCharacteristic Frequencies of Organic Molecules. Academic Press, Inc,San Diego Calif.). The band at 889 cm⁻¹ is representative of methylenerocking, and the 1294 cm⁻¹ band is typical of methylene twisting. TheC—C skeletal stretching vibrations appeared between 1060 cm⁻¹-1130 cm⁻¹.In this region, information about the conformation of the carbon chaincan also be obtained. The bands observed around 1060 cm⁻¹ and 1130 cm⁻¹are characteristic of trans bonded carbon and the band observed around1100 cm⁻¹ denotes the vibration of a gauche bonded chain. The analysisof the spectra obtained for these LPA samples compared well to Ramanspectra of various lipids that have been previously analyzed (Dai et al(2005) Colloids and Surfaces B 42: 21-28; Krafft et al. (2005)Spectrochim. Acta (61): 1529-1535; Saint-Pierre Chazelet et al. (1994)Thin Solid Films 244: 852-856; and Suh (1992) Chem. Phys. Lett. 193:327-330). Very strong similarities between the spectra of these two LPAmolecules were noted, in particular the 889 cm⁻¹, 1294 cm⁻¹, 1060 cm⁻¹,and 1128 cm⁻¹ bands that they commonly share. Fortunately, the 16:0 LPAwas distinguishable from the 18:0 LPA by the shift of the C—C vibrationof the gauche-bonded chain from 1097 cm⁻¹ for 16:0 LPA to 1101 cm⁻¹ for18:0 LPA.

FIG. 17 presents the SERS spectra of the two LPA samples that were driedon the silver nanoparticles after subtracting the background signal ofthe dried nanoparticle solution. Attempts to obtain the Raman spectra ofdried samples of LPA solutions without any silver present resulted in nosignal, indicating that the presence of the silver enhanced the LPARaman signal and made the detection of low concentrations possible. Asexpected, the bands of the acyl chain were enhanced using this techniquewith little to no shift from their bulk Raman positions. The SERS signalof the 16:0 LPA maintained its peaks of 889 cm⁻¹, 1097 cm⁻¹, 1128 cm⁻¹,and 1294 cm⁻¹, while 18:0 LPA maintained its peaks of 889 cm⁻¹, 1101cm⁻¹, 1128 cm⁻, and 1294 cm⁻¹. FIG. 18 shows the region between 1050cm⁻¹-1130 cm⁻¹, demonstrating the ability of this procedure to detectthe gauche peak that allows the acyl chains of the LPA samples to bedistinguished from each other is clearly observed.

In SERS, the relative enhancement of a given mode implies the preferredorientation of the adsorbate to the surface of the metal. Typically,enhancement of a given mode is best when it is closest to the surface ofthe metal. Comparing the band intensities of the SERS spectrum of eitherLPA sample to its respective bulk spectrum shows that the two are quitesimilar (see Table 1). In other words, the intensity distribution of theSERS modes of 16:0 LPA exhibited a similar pattern of its bulk spectrum.The same pattern was observed for the 18:0 samples. This implied that nostrong attraction was occurring between the nanoparticle surface andfunctional groups on the molecule to promote a specific orientation ofthe adsorbate. Also, with any strong surface interaction between theadsorbate and metal present, one would expect a shift in some bands ofthe SERS spectrum compared to the bulk Raman spectrum due to vibrationalhindrance that would result from the adsorbate-metal surfaceinteraction. This phenomenon was not observed in the LPA SERS spectra.The conclusion that no strong interaction was present between the metalsurface and adsorbate could also be made from the fact that no immediateSERS was observed for mixed solutions of silver and LPA. The interactionbetween LPA and the nanoparticle surface was only strong enough for SERSto be observed when the molecule was dried on top of the silver.

TABLE 1 Comparison of the intensity of the assigned vibrational modes ofthe Raman spectrum of the bulk samples of LPA with its SER signal (W =weak, M = medium, S = strong, VS = very strong) 16:0 18:0 18:0 Mode(cm⁻¹) Assignment LPA 16:0 SERS LPA SERS  889 CH₂ rock M M M M 1060 C—Cvib (trans) S — S — 1097 or 1101 C—C vib (gauche) M M M M 1128 C—C vib(trans) S S VS VS 1294 CH₂ (twist) S W VS M

Experiments to improve the sensitivity of this technique in terms of itsability to detect lower concentrations of various LPA in mixed samplesalong with actual samples of plasma/blood where other lysophospholipidsbesides LPA are present are performed. Some preliminary experimentsusing a prepared sample of mixed 16:0 and 18:0 LPA solutions had shownthat this technique was able to distinguish the two different LPAmolecules from each other in millimolar concentrations. However, inorder to apply SERS for practical LPA detection, this technique shoulddetect in micromolar quantities. As the surface interaction between themolecule and the nanoparticles play an important role in the effectiveenhancement of this technique, experiments are conducted with othermetal nanoparticles capped with various surface agents that may inducestronger interactions between the acyl chain of the adsorbate and metal.We also can apply SERS detection of LPA using different shaped metalnanoparticles, as non-spherical particles with sharp edges or cornersshow stronger SERS activities than spherical particles (Schatz (1984)Acc. Chem. Res. 17: 370-376; Gersten (1980) J. Chem. Phys. 72:5779-5780).

Example VI Detection of Ab-GNP Binding Interaction Using a Secondary Ab

The effect of binding an antigen to its antibody is observed by takingthe Raman spectrum of the antibody before and after exposure to theantigen through the use of SERS. To study the applicability of thismethod, a primary antibody (SC2020, Santa Cruz Biotechnology Santa CruzCalif.) and a secondary antibody (SC1616, Santa Cruz Biotechnology SantaCruz Calif.) were used. SC2020 was obtained at a concentration of 400μg/ml and diluted by a factor of two with 20 mM HEPES buffer (pH 7.4).This solution was mixed equal volume with a GNP solution that was alsodiluted by a factor of two with 20 mM HEPES buffer. After twenty minutesof interaction, a SERS spectrum was obtained. An equal amount of SC1616was added to the system and the SERS spectrum was obtained again. Thebinding of the secondary antibody (SC1616) to the primary antibody(SC2020) caused the SERS intensity of the secondary antibody to increaseby 20-50%. This method provides an indirect means of detecting antigensin a system.

Example VII Detection of Tumour-Antigens in Bodily Fluids

A murine monoclonal antibody raised against the CA125 ovarian cancermarker (OC125; Bast et al. (1981) J. Clin. Invest. 68: 1331-1337; Cat.No. AB19551, AbCam Ltd., Cambridge, UK) is incubated at a finalconcentration of 100 μg/ml in HEPES buffer (pH 7.4) with GNA as preparedabove at a final concentration of 1 mg/ml for twenty minutes at ambienttemperature. The mixture is then washed four times with excess samplebuffer, then stored at 4° C. until use. A fraction is subjected to SERSto obtain baseline values.

Fluid samples from individuals with diagnosed ovarian cancer areincubated with SQD in the presence of a conjugating agent and linkermolecule for 20 minutes at ambient temperature. The mixture is washedfour times and resuspended in HEPES buffer (pH 7.4) to produce SQD-Agconjugate. A fraction is subjected to SQD luminescence to obtainbaseline values.

The SQD-Ag conjugate is added to OC125-GNA mixture in HEPES incubationmedium (pH 7.4) at ambient temperature for 8 hours. Control samples arefrom individuals without diagnosed disease or disorders. The samples arethen washed four times with incubation medium, resuspended in samplebuffer, and then divided into two fractions. One fraction is subjectedto SQD luminescence. The other fraction is subjected to SERS. Baselinevalues obtained earlier are then compared with the values obtained underexperimental conditions.

Example VIII Production of Antigen Specific Antibodies

Antigen substantially purified using polyacrylamide gel electrophoresis(PAGE; see, for example, Harrington (1990) Methods Enzymol. 182:488-495) or other purification techniques is used to immunize rabbitsand to produce antibodies using standard protocols. The antigen aminoacid sequence is analyzed using DNASTAR software (DNASTAR Inc., MadisonWis.) to determine regions of high immunogenicity, and a correspondingoligopeptide is synthesized and used to raise antibodies by means knownto those of skill in the art. Methods for selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regionsare well described in the art. (See, for example, Ausubel et al. supra,chapter 11.)

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431 Ausing Fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis, Mo.)by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester toincrease immunogenicity. (See, for example, Ausubel et al. supra.)Rabbits are immunized with the oligopeptide-KLH complex in completeFreund's adjuvant. Resulting antisera are tested for antipeptideactivity, for example, by binding the peptide to plastic, blocking with1% BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG. In the alternative, a non-peptideantigen is used and is conjugated to KLH.

Example IX Purification of Naturally Occurring Antigen Using SpecificAntibodies

Naturally occurring or recombinant antigen is substantially purified byimmunoaffinity chromatography using antibodies specific for the antigen.An immunoaffinity column is constructed by covalently couplinganti-antigen antibody to an activated chromatographic resin, such asCNBr-activated Sepharose (Pharmacia & Upjohn, Kalamazoo Mich.). Afterthe coupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing antigen are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of antigen (for example, high ionic strength buffers in thepresence of detergent). The column is eluted under conditions thatdisrupt antibody/antigen binding (for example, a buffer of pH 2 to pH 3,or a high concentration of a chaotrope, such as urea or thiocyanateion), and antigen is collected.

Example X Identification of Molecules That Interact with Antigen

Antigen, or biologically active fragments thereof, are labeled with[¹²⁵I] Bolton-Hunter reagent. (See, for example, Bolton and Hunter(1973) Biochem. J. 133: 529-539.) Candidate molecules previously arrayedin the wells of a multi-well plate are incubated with the labeledantigen, washed, and any wells with labeled antigen complex are assayed.Data obtained using different concentrations of antigen are used tocalculate values for the number, affinity, and association of antigenwith the candidate molecules.

Example XI SERS Sensor Based on D-Shaped Fiber

Fiber surface enhanced Raman scattering (SERS) sensors show greatpotential for in vivo and in vitro detection, however, current probesbased on end polished fibers suffer from small signal due to their smallactive region. To overcome this, we propose and demonstrate a D-shapedfiber configuration to increase the detection area. Initial modeling hasshown that most of the light can be absorbed by the SERS active layercoated on the polished fiber surface. Several orders of magnitudeincrease in surface area leads to substantially more detectable Ramanscattered photons than those in end tip configurations. The SERS sensorbased on D-shaped fibers has been demonstrated, for the first time, withexcellent results using rhodamine 6G.

The majority of previous work in this area has utilized colloidalsolutions or single use films to great effect, however, for practicalapplications the substrate must be portable, reusable, and robust (seeSchwarzberg et al. (2004) supra; Tao et al. (2003) Nano Letters 3: 1229;van Duyne (2002) Abstracts of Papers of the American Chemical Society223: 3; Sagmulleret al. (2003) J. Mol. Struct. 661: 279; Michaels et al.(2000) J. Phys. Chem. B 104: 11965; and Jana (2003) Analyst 128: 954).Recently, significant advancements have been made to this end. End tipfiber optic SERS probes have been shown to produce excellent resultswith high stability and portability, where a fiber with a flat, angled,or tapered tip was modified with silver island films, roughened silverfilms, or silver film over nanospheres to produce the SERS substrate(see Stokes et al. (2004) Applied Spectroscopy 58: 292; Stokes andVo-Dinh (2000) Sensors and Actuators B-Chemical 69: 28; Mullen andCarron (1991) Anal. Chem. 63: 2196; Gessner et al. (2004) Analyst 129:1193; Viets and Hill (1998) Sensors and Actuators B-Chemical 51: 92;Viets and Hill (2000) J. Raman Spectrosc. 31: 625; and Viets and Hill(2001) J. Mol. Struct. 565: 515). This configuration is highlyadvantageous, being reproducible, facile to fabricate, and inexpensive.The main limitation is the small number of SERS particles on the activefiber region, requiring high laser intensity and long integration timesto attain reasonable SERS spectrum.

To overcome this hurdle, we chose to use a D-shaped fiber (DSF)configuration, so named because of the cross sectional D-shape formed byside polishing the fiber (see FIG. 19). Light can be coupled out of thepolished fiber into silver or gold nanostructured films coated onto thepolished surface, which can potentially increase the active surface areaby several orders of magnitude. DSF has been used in a variety ofapplications including humidity, temperature, strain sensing,communication, optical switching, attenuators, and polarizers, but neverfor SERS detection, to our knowledge (see McCallion and M. Shimazu(1998) Laser Focus World 34: S19; Sohn et al. (2002) Sensors andActuators A-Physical 101: 137; Alvarez-Herrero et al. (2000) IEEEPhotonics Technol. Lett. 12: 1043; and Gu et al. (2003a) J. OpticsA-Pure Appl. Optics 5: S420; Gu et al. (2003b), Optical Materials 23:219).

Procedures for polishing the DSF have been described previously (Xu(2003) In-line fiber optical components for telecommunication inelectrical engineering, University of California, Santa Cruz, SantaCruz, Calif.). The surface of the flat polished plane was purposely leftrough to facilitate nanoparticle binding and increase active surfacearea. Silver nanoparticles were synthesized by the method of Lee andMeisel (Lee and Meisel (1982) J. Phys. Chem. 86: 3391).

The SERS fibers were created by mixing 20 μl of silver nanoparticleswith 5 μl of 0.1 mM rhodamine 6G (R6G) and drying a drop of thissolution on the fiber under ambient conditions. SERS experiments werethen performed on these devices in two configurations. A 780 nm diodelaser is either coupled into the fiber (FIG. 19) or into a Renishawmicro-Raman spectrometer with a 20× objective. In both configurationsthe micro-Raman spectrometer collects the scattered light from the DSF.Samples were nominally integrated for 40 seconds for one scan with alaser intensity of ˜3 mW.

On a highly polished DSF, little or no light would be coupled out of thefiber core. When the surface is roughened and coated with a layer ofmetal such as silver nanoparticles, light can be coupled into thecoating layer to induce SERS. The amount of light coupled into the filmis determined by several parameters including polishing depth, filmthickness and density, and nature of the metal. The proper polishingdepth should be determined by calculation, however, drop-casting asilver nanoparticle film onto the DSF ensures a thick coating which willcouple the maximum amount of light from the fiber core into the SERSsubstrate.

To determine how the light will be coupled out of the fiber into theSERS substrate, we simulated light propagation inside a D-shaped fibercovered with a solid silver film as the colloidal film, using FIMMWAVEsoftware. Various film thickness, polishing depth, refractive index, andabsorption constants were used in our simulation. We found that theamount of light being coupled into the metal layer strongly depends onthe refractive index and the absorption constant of the metal. As anexample, FIG. 20 shows the simulation result of a Corning 28e fibercovered with a silver nanoparticle aggregate layer. The plot in FIG. 20shows the intensity distribution across the polished surface (top viewillustrated). The fiber parameters were: core diameter 48 μm,n_(core)=1.450, cladding diameter 125 μm, n_(clad)=1.4447, and thepolished surface cuts through the center of the fiber core. The metalfilm parameters were: thickness=0.1 μm, n_(Ag)=0.147, α_(Ag)=82000/cm.Here a large absorption constant was chosen because of the resonantabsorption of the Ag nanoparticle aggregates at 780 nm wavelength. Withthis choice of parameters, as light propagated through the side-polishedsection (from left to right in FIG. 20), the total intensity decreaseddue to coupling into the metal film and metal absorption, with only 30%remaining in the core at the end of the 1 cm side-polished fibersegment. The absorbed light activated SERS scattering over the entire 1cm×8 μm=80,000 μm² surface region above the fiber core, as opposed tothe 50 μm² of an end polished fiber of the same kind. As shown in theinset of FIG. 20, within a cross-section perpendicular to the fiberaxis, light was mostly confined in the core region. Utilizing the DSFwould increase the SERS active area, and consequently Raman scatteredlight, by as much as three orders of magnitude since light can becoupled into the metal layer over a substantial distance.

Having confirmed the viability of the concept, SERS experiments wereconducted using rhodamine 6G as a reference molecule resulting inexcellent SERS data. With the laser coupled through the microscope andaligned perpendicular to the DSF surface, large enhancements areobserved in the Raman modes of rhodamine 6G (FIG. 21, B). As expected,all peaks observed in the SERS spectrum were consistent with previousSERS results of rhodamine 6G (see Hildebrandt and Stockburger (1984) J.Phys. Chem. 88: 5935). More interestingly, instead of coupling lightthrough the Raman optical microscope, light coupled into the fiber fromthe end of the fiber (see FIG. 19) produced similarly intense signalsemitted and detected from the DSF surface (FIG. 21, A). In this case,light coupled from the core of the fiber into the silver nanoparticlefilm on the surface activates the plasmon mode and induced SERS of R6Gon the film. It is important to note that after background subtractionthe SERS spectra measured from both configurations were completelyconsistent. While peak intensities vary slightly, peak positionsremained nearly constant. This indicated that the scattering mechanismwas the same, independent of the excitation configuration.

One should be careful when comparing these spectra since theirexcitation intensities are different. The intensity of illuminationthrough the microscope objective was high and confined to a small areadue to the strong focusing of the excitation beam. The light coupledthrough the fiber had lower intensity over a larger area. Excitationintensity and surface area affect the scattering strength. Thevariations of peak intensities in the two spectra collected using twodifferent illumination configurations could be partly due to thedifference in excitation intensity.

In conclusion, we proposed and demonstrated a SERS sensor based onD-shaped fibers. Initial modeling had shown that as much as 70% of lightcoupled into the fiber may be absorbed into the SERS active layer acrossmuch of the 1 cm×8 μm surface, yielding an 80,000 μm² active region fora D-shaped fiber as compared to ˜50 μm² of an end polished fiber of thesame kind. This leads to as much as three orders of magnitude increasein Raman scattered photons compared to end tip fiber probes. The devicewas tested with rhodamine 6G with light directly illuminating the fibersurface and coupled through the fiber. Both configurations yieldexcellent and consistent SERS spectra of R6G. The experimental results,in conjunction with the theoretical modeling, demonstrated successfullythat D-shaped fibers can serve as a convenient platform for SERS sensorsthat can potentially provide extremely high sensitivity and molecularspecificity.

Those skilled in the art will appreciate that various adaptations andmodifications of the just-described embodiments can be configuredwithout departing from the scope and spirit of the invention. Othersuitable techniques and methods known in the art can be applied innumerous specific modalities by one skilled in the art and in light ofthe description of the present invention described herein. Therefore, itis to be understood that the invention can be practiced other than asspecifically described herein. The above description is intended to beillustrative, and not restrictive. Many other embodiments will beapparent to those of skill in the art upon reviewing the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A chemical sensor comprising a plurality of particles, each particlecomprising: a core, a shell having at least one surface and havingcontact with the core and wherein the shell comprises a sulfur-oxygenmolecular species, and wherein the particle has been selectively sizedusing a notch filter and electromagnetic radiation, the electromagneticradiation having a spectral wavelength of between about 350 nm and about1075 nm.
 2. The chemical sensor of claim 1 wherein the core comprises ametal selected from the group consisting of gold, silver, platinum,copper, aluminum, palladium, cadmium, iridium, and rhodium.
 3. Thechemical sensor of claim 1 wherein the core comprises gold.
 4. Thechemical sensor of claim 1 wherein the shell that further comprises alinker molecule, the linker molecule selected from the group consistingof a thiol group, a sulphide group, a phosphate group, a sulphate group,a cyano group, a piperidine group, an Fmoc group, and a Boc group. 5.The chemical sensor of claim 1 wherein the electromagnetic radiation hasa spectral wavelength of between about 350 nm and about 650 nm andbetween about 950 nm and about 1075 nm.
 6. The chemical sensor of claim1 wherein the electromagnetic radiation has a spectral wavelength ofbetween about 350 nm and about 775 nm and between about 875 nm and about1075 mn.
 7. The chemical sensor of claim 1 wherein the particle has asize in the range of about 60 and 200 nm.
 8. The chemical sensor ofclaim 1 further comprising a support.
 9. The chemical sensor of claim 8wherein the support comprises a medium that is permeable to an analyteof interest.
 10. The chemical sensor of claim 1 wherein the surface caninduce surface enhanced Raman scattering.
 11. The chemical sensor ofclaim 1 further comprising a detecting molecule, wherein the detectingmolecule is bound to the surface.
 12. The chemical sensor of claim 11wherein the detecting molecule is selected from the group consisting ofproteins, peptides, antibodies, antigens, nucleic acids, peptide nucleicacids, sugars, lipids, glycophosphoinositols, and lipopolysaccharides.13. The chemical sensor of claim 11 wherein the detecting molecule is anantibody.
 14. The chemical sensor of claim 11 wherein the detectingmolecule is an antigen.
 15. The chemical sensor of claim 1 furthercomprising a semiconductor quantum dot.
 16. The chemical sensor of claim15 wherein the semiconductor quantum dot further comprises a linkermolecule, the linker molecule selected from the group consisting of athiol group, a sulphide group, a phosphate group, a sulphate group, acyano group, a piperidine group, an Fmoc group, and a Boc group.
 17. Thechemical sensor of claim 15 wherein the semiconductor quantum dotfurther comprises a detecting molecule, wherein the detecting moleculeis bound to the semiconductor quantum dot.
 18. The chemical sensor ofclaim 15 wherein the detecting molecule is selected from the groupconsisting of proteins, peptides, antibodies, antigens, nucleic acids,peptide nucleic acids, sugars, lipids, glycophosphoinositols, andlipopolysaccharides.
 19. The chemical sensor of claim 15 wherein thedetecting molecule is an antibody.
 20. The chemical sensor of claim 15wherein the detecting molecule is an antigen.
 21. The chemical sensor ofclaim 20 wherein the detecting molecule is an antigen that binds to anovarian cancer marker antibody with an affinity (K_(a)) of at least 10⁶l/mole.
 22. The chemical sensor of claim 21 wherein the K_(a) is atleast 10⁸ l/mole.
 23. A method for detecting an analyte in a sampleusing a chemical sensor, the method comprising the steps of: i)providing a sample; ii) providing a semiconductor quantum dot comprisinga linker molecule (LM-SQD) iii) conjugating the analyte in the samplewith the LM-SQD thereby producing an analyte-LM-SQD conjugate; iv)providing the chemical sensor of claim 11; v) incubating theanalyte-LM-SQD conjugate with the chemical sensor for a predeterminedtime period; and vi) measuring the extent of binding between theanalyte-LM-SQD conjugate and the chemical sensor; thereby detecting theanalyte in the sample.
 24. The method of claim 23 wherein the analyte isan ovarian cancer marker antibody.
 25. The method of claim 23 whereinthe detecting molecule in the chemical sensor is an antigen that bindsto an ovarian cancer marker antibody with an affinity (K_(a)) of atleast 10⁶ l/mole.
 26. The method of claim 25 wherein the K_(a) is atleast 10⁸ l/mole.
 27. An optical communications device comprising afiber and the chemical sensor of claim
 1. 28. The optical communicationsdevice of claim 27 wherein the fiber is selected from the groupconsisting of ceramics, glasses, and polymers.
 29. The opticalcommunications device of claim 27 wherein the fiber cross-section isD-shaped.
 30. The optical communications device of claim 27 wherein thechemical sensor is disposed upon a surface of the fiber.